A model for studying leukemia cell differentiation in vitro has been constructed. The HL-60 cell line, established from human promyelocytic leukemia, was used to obtained a variant cell line, 1F10, which, contrary to the original HL-60 cell line, needs two inducers to differentiate into monocyte-macrophage-like cells. The two inducers, when used separately, allow us to define discrete complementary steps in the differentiation pathway. To identify genes that are regulated at discrete steps in HL-60 differentiation, cDNA subtraction was applied to the IF 10 system. By this method, we have isolated a cDNA clone encoding a novel serine protease, myeloblastin. Myeloblastin mRNA is down-regulated by granulocytic and monocytic inducers of HL-60 differentiation. In the absence of inducer, myeloblastin mRNA can be regulated by serum. We have used an antisense oligodeoxynucleotide to inhibit myeloblastin expression. This inhibition resulted in proliferation arrest and differentiation of the leukemic cells. Our project is to investigate the transcriptional mechanisms involved in the regulation of the myeloblastin gene expression. These transcriptional mechanisms may be critical to the understanding of leukemogenesis and normal differentiation.